Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila

  1. Zaijun Ma
  2. Hui Wang
  3. Yuping Cai
  4. Han Wang
  5. Kongyan Niu
  6. Xiaofen Wu
  7. Huanhuan Ma
  8. Yun Yang
  9. Wenhua Tong
  10. Feng Liu
  11. Zhandong Liu
  12. Yaoyang Zhang
  13. Rui Liu
  14. Zheng-Jiang Zhu  Is a corresponding author
  15. Nan Liu  Is a corresponding author
  1. Shanghai Institute of Organic Chemistry, China
  2. University of Chinese Academy of Sciences, China
  3. State Key Laboratory of Medical Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, China
  4. Texas Children’s Hospital, United States
  5. Baylor College of Medicine, United States
  6. Singlera Genomics, China
9 figures, 1 table and 6 additional files

Figures

Figure 1 with 1 supplement
Adult-onset fidelity loss results in epigenetic drift of H3K27me3.

(A) H3K27me3 peak profiles are comparable between young and aged animals. A scatter plot (top panel) of 331 peaks and a genome browser mini-view (bottom, left panel) illustrated that H3K27me3 had …

https://doi.org/10.7554/eLife.35368.002
Figure 1—figure supplement 1
Global occupancy of H3K27me3 modification is preserved with age.

(A) Western blot (top) and quantification (bottom) show an increase of H3K27me3 with age in muscle tissues. (mean ±SD of three biological repeats; student t-test). Genotype: 5905. (B) Quantitative …

https://doi.org/10.7554/eLife.35368.003
Figure 2 with 1 supplement
PRCs-deficient animals have extended lifespan.

(A) PRCs-deficient animals have extended lifespan. H3K27me3 western blot (left), H3K27me3 quantification (middle), and lifespan curve (right) for PRC single mutants. PRC2 heterozygous mutants of …

https://doi.org/10.7554/eLife.35368.004
Figure 2—figure supplement 1
PRCs-deficient animals have extended lifespan.

(A) PRCs-deficient animals have extended lifespan. PRC2 heterozygous mutants of indicated genotype reduce H3K27me3 levels and extend lifespan (top of the dashed line); PRC1 heterozygous mutants of …

https://doi.org/10.7554/eLife.35368.005
Figure 3 with 1 supplement
Long-lived PRC2 mutants diminish the epigenetic drift of H3K27me3 during aging.

(A) Circos plot of the H3K27me3 epigenome illustrates peak profiles that are highly preserved with age and in PRC2 mutants. Black boxes and lines (innermost circle) represented common peak regions, …

https://doi.org/10.7554/eLife.35368.006
Figure 3—figure supplement 1
Long-lived PRC2 mutants diminish the epigenetic drift of H3K27me3 during aging.

(A) Relative quantification of different histone marks between WT and PRC2 mutants. Only H3K27me2/3 were selectively reduced in PRC2 mutants. Western blot was from head tissues of 3d-old male flies. …

https://doi.org/10.7554/eLife.35368.007
Figure 4 with 1 supplement
Epigenetic drifting of H3K27me3 occurs with age in head.

(A) H3K27me3 modification increases with age in head. Scatter plot showed H3K27me3 levels for protein-coding genes (transcriptional start site to transcriptional termination sites as annotated in …

https://doi.org/10.7554/eLife.35368.008
Figure 4—figure supplement 1
Global occupancy of H3K27me3 modification in head tissues with age and in PRC2-deficiency.

(A) Western blot (top) and quantification (bottom) show an increase of H3K27me3 with age in head tissues. (mean ±SD of three biological repeats; student t-test). Genotype: 5905. (B) The distribution …

https://doi.org/10.7554/eLife.35368.009
Figure 5 with 1 supplement
Transcriptomics links H3K27me3 dynamics to the regulation of glycolytic genes.

(A) Venn diagram shows genes commonly changed in PRC2 single mutants with indicated genotype (left panel). Differential expression genes were computed using DESeq based on normalized count from …

https://doi.org/10.7554/eLife.35368.010
Figure 5—figure supplement 1
Transcriptomics links H3K27me3 dynamics to the regulation of glycolytic genes.

(A) Number of genes upregulated in PRC2 mutants with indicated genotype. Differential expression genes were computed using DESeq based on normalized count from three biological replicates (cutoff: p<…

https://doi.org/10.7554/eLife.35368.011
Figure 6 with 2 supplements
Metabolomics shows the effect of PRC2 mutants in reversing glycolytic decline in aging.

(A) Untargeted metabolics identifies that metabolites of the glycolytic pathway (red) are decreased with age. For each metabolite, the median from 10 biological repeats was used to calculate the …

https://doi.org/10.7554/eLife.35368.012
Figure 6—figure supplement 1
Untargeted metabolomics.

(A) Heatmap of metabolites exhibiting differential levels of 3d, 30d WT and 30d PRC2 mutants. Data normalization using support vector regression algorithm. Metabolomics was from head tissues of 3d- …

https://doi.org/10.7554/eLife.35368.013
Figure 6—figure supplement 2
Metabolic glucose flux experiment.

(A) Schematic representation of the glucose flux experiment in adult aging flies. Briefly, flies at 3d (young) and 25d (aged) were switched from 12C-glucose to 13C-glucose food for a fixed 5days, …

https://doi.org/10.7554/eLife.35368.014
PRC2 mutants couple enhanced glycolysis with improved adult fitness.

(A) Glucose content shows a slight increase in PRC2 mutants, but this increase is not statistically significant (left panel). Pyruvate, one key end product of glycolysis, is increased (right panel). …

https://doi.org/10.7554/eLife.35368.015
Figure 8 with 1 supplement
Perturbing glycolysis diminishes longevity benefits in PRC2 mutants.

(A) Tpi deficiency significantly diminishes the longevity phenotype of PRC2 trans-heterozygous double mutants. (for lifespan assay: 25°C; n > 200 per genotype; log-rank test). Genotypes: Pclc421/+; S…

https://doi.org/10.7554/eLife.35368.016
Figure 8—figure supplement 1
Tpi- and Pgi-deficient flies have normal adult phenotypes.

(A)-(D) and (F)-(I) Assessments of adult lifespan (A) and (F), oxidation test (B) and (G), climbing ability (C) and (H), and the levels of ATP and the ratio of NADH/NAD+ (D) and (I), show no …

https://doi.org/10.7554/eLife.35368.017
Figure 9 with 1 supplement
Transgenic increase of glycolytic genes suffices to elevate glycolysis and healthy lifespan.

(A) Tpi protein western blot (top) and quantification (bottom) shows a decrease with age. (mean ±SD of three biological repeats; student t-test). Western blot was from 3d- and 30d-old male flies. …

https://doi.org/10.7554/eLife.35368.018
Figure 9—figure supplement 1
Genomic transgenes for Tpi and Pgi.

(A) and (B) Genomic structure and gene isoforms were shown according to the Flybase annotation. Color codes represented ATG (the translation start site), HA tag, CDS (coding sequence), 5’UTR, and …

https://doi.org/10.7554/eLife.35368.019

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Cell line
(D. melanogaster)
S2R+This paperFLYB:FBtc0000150
AntibodyH3K27me3
(rabbit polyclonal)
MilliporeCat#07–449;
RRID:AB_310624
AntibodyH3K27me2
(rabbit polyclonal)
AbcamCat#ab24684;
RRID:AB_448222
AntibodyH3K27me
(rabbit polyclonal)
MilliporeCat#07–448
AntibodyH3K27ac
(rabbit polyclonal)
AbcamCat#ab4729;
RRID:AB_2118291
AntibodyH3K9me3
(rabbit polyclonal)
AbcamCat#ab8898;
RRID:AB_306848
AntibodyH3K9ac
(rabbit polyclonal)
MilliporeCat#06–942;
RRID:AB_310308
AntibodyH3K23ac
(rabbit polyclonal)
MilliporeCat#07–355;
RRID:AB_310546
AntibodyH3K4me3
(rabbit polyclonal)
MilliporeCat#07–473;
RRID:AB_1977252
AntibodyH3K4me2
(rabbit polyclonal)
MilliporeCat#07–030;
RRID:AB_310342
AntibodyH3K4me
(rabbit polyclonal)
MilliporeCat#07–436;
RRID:AB_310614
AntibodyH3K36ac
(rabbit polyclonal)
MilliporeCat#07–540
AntibodyH3K36me3
(rabbit polyclonal)
AbcamCat#ab9050;
RRID:AB_306966
AntibodyH3K14ac
(rabbit polyclonal)
MilliporeCat#07–353;
RRID:AB_310545
AntibodyH3K18ac
(rabbit polyclonal)
AbcamCat#ab1191;
RRID:AB_298692
AntibodyH4K20me3
(rabbit polyclonal)
AbcamCat#ab9053
AntibodyH3 (goat polyclonal)AbcamCat#Ab12079
AntibodyHA (rabbit monoclonal)Cell signaling technologyCat#3724
AntibodyTubulin
(rabbit polyclonal)
Medical and biological
laboratories co
Cat#PM054
AntibodyAnti-Rabbit IgGSigmaCat#A9169
AntibodyAnti-Mouse IgGSigmaCat#A4416
AntibodyAnti-Goat IgGAbcamCat#ab6471
Recombinant DNA
reagent
pBID UAS vector: HA-TpiAddgene35198
Recombinant DNA
reagent
pBID UAS vector:
HA-Pgi
Addgene35198
Recombinant DNA
reagent
pBID vector:
HA-Tpi genomic
Addgene35190
Recombinant DNA
reagent
pBID vector:
HA-Pgi genomic
Addgene35190
Recombinant DNA
reagent
pU6b-sgRNARen et al., 2013N/A
Commercial assay or kitTURBO DNA-free kitThermoFisherCat#AM1907
Commercial assay or kitSuperSignal West Pico
Chemilluminescent
Subtrate
ThermoFisherCat#34078
Commercial assay or kitNuclear-Cytosol
Extraction Kit
AogmaCat#9988
Commercial assay or kitSYBR Select Master MixThermoFisherCat#26161
Commercial assay or kitSuperScript III First-strand
synthesis system for RT-PCR
ThermoFisherCat#18080–051
Commercial assay or kitChIP-Grade Protein A/G
Plus Agarose
ThermoFisherCat#26161
Commercial assay or kitQIAquick PCR Purification KitQIAGENCat#28106
Commercial assay or kitQubit dsDNA HS Assay KitThermoFisherCat#Q32851
Commercial assay or kitHigh Sentivity DNA Analysis KitsAgilent TechnologiesCat#5067–4626
Commercial assay or kitNEBNext DNA Library Prep KitNew England BiolabsCat#E7370L
Commercial assay or kitNEBNext Poly(A) mRNA
Magnetic Isolation Module
New England BiolabsCat#E7490L
Commercial assay or kitNEBNext Multiplex Oligos
for Illumina
New England BiolabsCat#E7600S
Commercial assay or kitNEBNext Ultra RNA library
Prep Kit for Illumina
New England BiolabsCat#E7530L
Commercial assay or kitRNase A/T1 MixThermoFisherCat#EN0551
Commercial assay or kitGlucose (HK) Assay KitSigmaCat#GAHK20
Commercial assay or kitNAD/NADH Quantitation KitSigmaCat#MAK037
Commercial assay or kitNADP/NADPH
Quantitation Kit
SigmaCat#MAK038
Commercial assay or kitENLITEN ATP Assay SystemPromegaCat#FF2000
Commercial assay or kitPyruvate Assay KitSigmaCat#: MAK071
Commercial assay or kitGSH/GSSG Ratio
Detection Assay
AbcamCat#ab13881
Commercial assay or kitAmicon Ultra 0.5 mL
centrifugal filters
SigmaCat#Z677108-96EA
Chemical compound,
drug
Tris-buffer (1 mol/L, pH8.5)Sangon BiotechCat#SD8141
Chemical compound,
drug
TRIZol RreagentThermoFisherCat#15596018
Chemical compound,
drug
ChloroformSinopharm Chemical ReagentCat#10006818
 Chemical compound,
drug
IsopropanolSinopharm Chemical ReagentCat#10006818
Chemical compound,
drug
DEPC-treated waterInvitrogenCat#46–2224
Chemical compound,
drug
NuPAGE 12% Bis-Tris GelThermoFisherCat#NP0342BOX
Chemical compound,
drug
PageRuler Prestained
Protein Ladder
ThermoFisherCat#26616
Chemical compound,
drug
NuPAGE MOPS SDS
Running Buffer
ThermoFisherCat#NP0001
Chemical compound,
drug
Immobilon Transfer
Membranes
MilliporeCat#: IPVH00010
Chemical compound,
drug
GlycinebeyotimeCat#ST085-1000G
Chemical compound,
drug
20 X PBSSangon BiotechCat#B548117-0500
Chemical compound,
drug
37% formaldehyde solutionSigmaCat#F1635
Chemical compound,
drug
Protease inhibitor
cocktail tablets
RocheCat#11697498001
Chemical compound,
drug
RIPA bufferSigmaCat#R-278–500 ML
Chemical compound,
drug
5M NaClThermoFisherCat#AM9760G
Chemical compound,
drug
0.5M EDTAThermoFisherCat#15575–038
Chemical compound,
drug
1M Tris-HClThermoFisherCat#15568–025
Chemical compound,
drug
Triton X-100SigmaCat#T8787-50ML
Chemical compound,
drug
10% SDSSangon BiotechCat#SD8118
Chemical compound,
drug
RNase A/T1 MixThermoFisherCat#EN0551
Chemical compound,
drug
Sodium dicarbonateSigmaCat#S5761
Chemical compound,
drug
Proteinase KSangon BiotechCat#A100706
Chemical compound,
drug
3M Sodium Acetate pH5.5ThermoFisherCat#AM9740
Chemical compound,
drug
Methyl viologen dichloride
hydrate
SigmaCat#856177
Chemical compound,
drug
Hydrogen peroxide 30%Sinopharm Chemical ReagentCat#10011208
Chemical compound,
drug
D(+)-ArabinoseSangon BiotechCat#A600071-0100
Chemical compound,
drug
D-glucoseSangon BiotechCat#A100188-0500
Chemical compound,
drug
Sodium hydroxide
solution
SigmaCat#72068
Chemical compound,
drug
MethanolHoneywellCat#LC230-2.5HC
Chemical compound,
drug
AcetonitrileMerckCat#1.00029.2500
Chemical compound,
drug
WaterHoneywellCat#LC365-2.5HC
Chemical compound,
drug
Ammonium acetateSigma-AldrichCat#73594–25 G-F
Chemical compound,
drug
Ammonium hydroxideSigma-AldrichCat#44273–100 mL-F
Chemical compound,
drug
Sequencing Grade
Modified Trypsin
PromegaCat#V5111
Chemical compound,
drug
Centrifugal Filters
Ultracel 30K
AmiconCat#UFC30SV00
Chemical compound,
drug
Water, LC/MSJ.T.BakerCat#9831–03
Chemical compound,
drug
Formic acid, eluent
additive for LC-MS
SigmaCat#56302–50 ml-f
Chemical compound,
drug
Acetonitrile,LC/MS,4LJ.T.BakerCat#9829–03
Chemical compound,
drug
Ammonium bicarbonateSigmaCat#09830–1 KG
Chemical compound,
drug
UreaSigmaCat#U5128-500G
Chemical compound,
drug
Trizma hydrochlorideSigmaCat#T5941-100G
Chemical compound,
drug
Ammonium formateFluckaCat#17843–50G
Chemical compound,
drug
Lys(6) SILAC yeastSilantesCat#234CXX-SYK6-509-01
Chemical compound,
drug
D-Glucose (U-13C6,99%)Cambridge Isotope Laboratories, Inc.Cat#110187-42-3
Software, algorithmBowtie2Langmead and Salzberg (2012)http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Software, algorithmSamtoolsLi et al. (2009)http://samtools.sourceforge.net/; RRID:SCR_002105
Software, algorithmdeepToolsRamírez et al., 2014https://deeptools.github.io/
Software, algorithmhomerHeinz et al., 2010http://homer.ucsd.edu/homer/ngs/index.html; RRID:SCR_010881
Software, algorithmbwtoolPohl and Beato, 2014https://users.dcc.uchile.cl/~peortega/bwtool/; RRID:SCR_003035
Software, algorithmGalaxyGrüning et al., 2017https://github.com/bgruening/galaxytools; RRID:SCR_006281
Software, algorithmCistromeLiu et al., 2011http://cistrome.dfci.harvard.edu/ap/; RRID:SCR_000242
Software, algorithmBEDToolsQuinlan and Hall, 2010http://bedtools.readthedocs.io/; RRID:SCR_006646
Software, algorithmJ-circosAn et al., 2015https://omictools.com/j-circos-tool; RRID:SCR_011798
Software, algorithmVENNYOliveros, 2007http://bioinfogp.cnb.csic.es/tools/venny/index.html
Software, algorithmRR Core Team, 2013https://www.r-project.org; RRID:SCR_001905
Software, algorithmDAVID Bioinformatics
Resources
Huang et al., 2009bhttps://david.ncifcrf.gov/; RRID:SCR_001881
Software, algorithmIGVRobinson et al., 2011http://software.broadinstitute.org/software/igv/; RRID:SCR_011793
Software, algorithmSTARDobin et al., 2013https://github.com/alexdobin/STAR; RRID:SCR_005622
Software, algorithmHTSeqAnders et al., 2015http://www-huber.embl.de/HTSeq/; RRID:SCR_005514
Software, algorithmDESeqAnders and Huber, 2010http://www-huber.embl.de/users/anders/DESeq/; RRID:SCR_000154
Software, algorithmWGCNALangfelder and Horvath, 2008https://labs.genetics.ucla.edu/horvath/htdocs/CoexpressionNetwork/Rpackages/WGCNA/; RRID:SCR_003302
Software, algorithmXCMSSmith et al., 2006http://www.bioconductor.org/packages/release/bioc/html/xcms.html; RRID:SCR_015538
Software, algorithmMetaboAnalyst 3.0Xia et al., 2015http://www.metaboanalyst.ca/; RRID:SCR_015539
Software, algorithmCAMERAKuhl et al. (2012)https://bioconductor.org/packages/release/bioc/html/CAMERA.html; RRID:SCR_011924
Software, algorithmPD1.4Thermohttps://www.thermofisher.com/order/catalog/product/IQLAAEGABSFAKJMAUH
Software, algorithmPathways to PCDL
(version B.07.00)
Agilent Technologies
Software, algorithmPCDL Manager
(version B.07.00)
Agilent Technologies
Software, algorithmProfinder
(version B.08.00)
Agilent Technologies
Software, algorithmPrismGraphPadv6; RRID:SCR_002798

Additional files

Supplementary file 1

Summary of H3K27me3 peak regions.

Summary of ChIP-seq experiments. Two criteria for peak calling: IP/input ≥ 2, signals spanning ≥3 kb. In total, 222 peak regions of H3K27me3 were reproducibly identified by four biological replicates at 3d. For each peak regions, information pertaining to chromosomal location and genes therein contained were given. ChIP-seq was using muscle tissues of 3d=old animals. Genotype: 5905.

https://doi.org/10.7554/eLife.35368.020
Supplementary file 2

Summary of CRISPR/Cas9-led gene mutagenesis and lifespan.

For each gene, two sgRNAs with indicated sequences, detailed deletion sites confirmed by Sanger sequencing, and PCR validation were shown. To name new CRISPR mutant, a superscript amended to the gene contained a letter c denoting CRISPR/Cas9-directed mutagenesis followed by the size of genomic deletion. Mutants were backcrossed (at least five generations) into 5905 (Flybase ID FBst0005905, w1118) to assure that phenotypes were not associated with any variation in background. Lifespan curves, together with 50% (median lifespan) and 10% survival data were listed. (for lifespan assay: 25°C; n ≥ 140 per genotype for curve; log-rank test).

https://doi.org/10.7554/eLife.35368.021
Supplementary file 3

Summary of transcriptomic analysis of PRC2 longevity mutants.

RNA-seq summary. This table summarizes five categories of genes and their transcriptional changes in long-lived PRC2 mutants of indicated genotype: (1) commonly upregulated and (2) downregulated genes in all PRC2 long-lived mutants; genes known to promote longevity upon upregulation, including (3) insulin pathway genes, (4) TOR signaling pathway genes, and (5) genes of miscellaneous pathways. RNA-seq was from muscle and head tissues of 30d-old male flies.

https://doi.org/10.7554/eLife.35368.022
Supplementary file 4

Summary of transcriptomic analysis of genes in glycolysis, citric acid cycle, pentose phosphorylation pathway, and oxidative phosphorylation.

This table summarizes metabolic genes and their transcriptional changes in long-lived PRC2 mutants of indicated genotype. RNA-seq was from head and muscle tissues of 3d- and 30d-old male flies.

https://doi.org/10.7554/eLife.35368.023
Supplementary file 5

Supplemental Materials and methods: Primer sequences used for the study.

https://doi.org/10.7554/eLife.35368.024
Transparent reporting form
https://doi.org/10.7554/eLife.35368.025

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